Interleukin-8 (IL-8) is a cytokine that activates and attracts neutrophils and attracts T-lymphocytes. IL-8 is released by several cell types including monocytes, macrophages, T-lymphocytes, fibroblasts, endothelial cells, and keratinocytes by an inflammatory stimulus. IL-8 is a member of the beta-thromboglobulin superfamily and structurally related to platelet factor 4.
IL-8 is a non-glycosylated protein of 8 kDa (72 amino acids) and is produced by processing of a precursor protein of 99 amino acids. The IL-8 protein contains four cysteine residues participating in disulfide bridges. The human IL-8 gene (SCYB8 ) has a length of 5.1 kb, contains four exons and maps to human chromosome 4q12-q21. The mRNA consists of a 101 base 5′ untranslated region, an open reading frame of 297 bases, and a long 3′ untranslated region of 1.2 kb. The 5′ flanking region of the IL-8 gene contains a number of cis-acting elements that are potential binding sites for nuclear factors. Some of the cis-acting elements are responsive to intrinsic regulators such as NFκB and AP-1, while others are pathogen-specific. The identities of the cis-acting elements are summarized as follows: (i)RSVRE, (binding site −162 to −132) is the newly discovered Respiratory Syncytial Virus Responsive Element, which is responsible for the intense IL-8-dependent pulmonary inflammation in RSV infection [Casola et al, J. Immunol., 164:5944-5951, (2000)]. (ii) IFNRF1,the Interferon Regulating Factor 1, binds to and further activates the RSVRE. [Casola et al, supra]. (iii) AP-1, (binding site: −126 to −120), is activated by the heterodimer of JunD/cFos, and is superactivated in cytomegalic virus (CMV) infection by the (iv) CMVRE1 (CMV Responsive Element). (v) NF-IL6 (binding site: −94 to −81) is activator of IL-8 transcription and is a possible overlapping site of interaction with C/EBPa (CCAAT box enhancer binding protein a). (vi) NFκB (binding site: −80 to −70) is activated by the family of NF B/Re1 transcription factors, and super-activated by CMV1E1. (vii) C/EBPa (binding site: −91 to −81) partially overlaps the NFκB site, and either activates or inhibits IL-8 transcription depending on the context [Victor et al, J. Trauma, 39:635-640, (1996); Stein et al., Mol. Cell. Biol., 13:7191-7198, (1993)]. (viii) GC (glucocorticoid receptor) binds to NFκB and inhibits transcription [Baldwin, Annu. Rev. Immunol., 14:649-681, (1996)]. (ix) OCT-1 is a homeo-domain factor that suppresses IL-8 transcription by acting on NF-IL6 [Wu et al, J. Biol. Chem., 272:2396-2403, (1997)]. (x) NF-AT (Nuclear Factor of Activated T cells) binds at or near the NFκB site and activates transcription [Roebuck, J. Interferon Cytokine Res., 19:429-438, (1999)]. (xi) TAX, coded by HTLV-1, binds to NFκB and promotes transcription [Suzuki et al, Oncogene, 9:3099-3105, (1994)]. (xii) NRF (binding site: partial overlap with NFκB) is the NFκB Repressing Factor which is principally responsible for basal silencing, but is also required for full IL-8 mRNA production [Nourbakhsh et al, J. Biol. Chem., 44:4501-4508, (2000)]. (xiii) TATA box (binding site: −20 to −13) binds TF-III and the TBP, and is absolutely required for IL-8 transcription.
IL-8 differs from all other cytokines in its ability to specifically activate neutrophil granulocytes. In neutrophils, IL-8 causes a transient increase in cytosolic calcium levels and the release of enzymes from granules. IL-8 also enhances the metabolism of reactive oxygen species and increases chemotaxis and the enhanced expression of adhesion molecules. A pre-activation by IL3 is required to render basophils and neutrophils susceptible to further activation by IL-8, and IL-8 alone does not release histamines. IL-8 actually inhibits histamine release from human basophils induced by histamine-releasing factors, CTAP-3 (connective tissue activating protein-3) and IL3.
L8 is chemotactic for all known types of migratory immune cells and inhibits the adhesion of leukocytes to activated endothelial cells, and therefore, possesses anti-inflammatory activities. IL-8 is a mitogen for epidermal cells and strongly binds to erythrocytes in vivo. This absorption may be of physiological importance in the regulation of inflammatory reactions since IL-8 bound to erythrocytes no longer activates neutrophils. Macrophage derived IL-8 supports angiogenesis and may play a role in angiogenes dependent disorders such as rheumatoid arthritis, tumor growth, and wound healing.
IL-8 expression and regulation have been associated a variety of disease conditions such as inflammatory bowel disease, atherosclerosis, and lung disorders. IL-8 has been specifically associated with cystic fibrosis (CF) because it is profoundly elevated in bronchoalveolar lavage fluids, sputum, and serum from CF patients [Dean et al, Pediatr. Res., 34:159-161, (1993) and Armstrong et al, Am J. Resnir. Crit. Care Med., 156:1197-1204, (1997)].
CF is the most common autosomal recessive lethal disease in the United States [Welsh et al, Cystic fibrosis in: The metabolic and molecular bases of inherited diseases (Scriver, C. L., Beaudet, A. L., Sly, W. S., and Valle, D. eds.) Seventh Ed. pp. 3799-3876, McGraw-Hill, New York. (1995)]. Approximately 5% of the population carries one mutant cystic fibrosis transmembrane conductance regulator (CFTR) gene [Rommens et al, Science, 245:1:1059-1065, (1989); Riordan et al, Science, 245:1:1066-1073, (1989); and Kerem et al, Science, 245:1:1073-1080, (1989)], and the disease occurs at a frequency of 1 in 2500 live births. Statistically, death occurs in the majority of patients by age 28. Respiratory difficulties and ensuing complications of inflammation and lung infection are directly responsible for the eventual death of over 90% of CF patents.
The CF lung has been described as microscopically normal at birth, with subtle abnormalities in mucus secretion appearing very early [Pilewski et al., Physiol. Rev., 79:5215-5255, (1999)]. Bacterial infection and objective evidence of inflammation occur at later times, with a clear temporal evolution of different principal bacterial pathogens. For example, Staphylococcus aureaus and Hemophilus influenzae take up residence in the CF airway early, the mean age of positive culture being 12.4 months [Abman et al, J. Pediatrics 119:211-217, (1991)]. By comparison, Pseudomonas aeruginosa infection follows at a substantially later time, the mean age of first positive culture being 20.8 months. Persistent colonization by P. aeruginosa characterizes the older CF patient, and profound, persistent cellular evidence of inflammation accompanies persistent infection as the patient approaches the terminal phases of the disease.
As the CF patient ages, the CF lung becomes characterized by elevated levels of white cells. These include polymorphonuclear leukocytes, macrophages, monocytes, lymphocytes and eosinophils. It is suggested that these cells are attracted from the circulation into the airway by the high levels of IL-8 and other pro-inflammatory factors such as IL1β, IL6, leukotriene B4, RANTES, and TNFα. These factors mark the character of the CF lumenal milieu [Bonfield et al, Am. J. Respir. Mol. Biol., 13:257-261, (1995); and Bonfield et al, Am. J. Respir. Mol. Biol., 125:2111-2118, (1995)], and among these factors, IL-8 ranks as the most prevalent and potent.